ABSTRACT We propose a mechanistic study of the biological responses of adult human bone marrow-derived mesenchymal stem cells (MSCs) to 3D substrate rigidity using hydrogel scaffolds of photocrosslink-controllable stiffness. Specifically, our focus is on the role of membrane cholesterol and caveolae subdomains, and focal adhesion signaling in these responses. The responses of MSCs to substrate rigidity, particularly in a 3D context, represent an important regulatory mechanism of their biological activities, of particular relevance to their application in tissue engineering and regeneration. We postulate here that these responses involve integrin-mediated focal adhesion signaling, and are potentially sensitive to cellular cholesterol homeostasis. We therefore propose to analyze the effects of the cholesterol/Caveolin-1 (Cav-1)/caveolae membrane system, which is known to regulate focal adhesion signaling, on MSC substrate rigidity responses, with special attention to differentiation. Information on the effects of this system on MSC behavior may be important in a wider context, if it is affected in vivo by cholesterol-modifying drugs, which are widely clinically prescribed and thus may influence outcomes of regenerative therapies. We hypothesize that elevated cell membrane levels of cholesterol/Cav-1/caveolae decrease MSC sensitivity to substrate stiffness through increasing integrin endocytosis and decreasing focal adhesion signaling. To test our hypothesis, we propose three specific aims in which we will use cholesterol depletion, cholesterol supplementation, Cav-1 knockdown, and pharmacological inhibitors, to study the roles of MSC membrane cholesterol, Cav-1, caveolae, and focal adhesion signaling in MSC rigidity sensing in our 3D experimental platform. AIM 1: Test the effects of perturbations in cholesterol/Cav-1/caveolae homeostasis on MSC membrane properties and adhesive characteristics. This will verify that manipulation of cholesterol/Cav- 1/caveolae impacts aspects of the MSC cell membrane important to substrate sensing, including integrin expression, activation and internalization, and the strength of cell adhesion to defined substrates. AIM 2: Test the responses of MSCs with and without perturbations in cellular cholesterol/Cav-1/caveolae homeostasis to varied stiffness in a 3D context. This will determine how MSCs respond to varied substrate rigidity in 3D, in terms of their morphology, substrate adhesion, cytoskeletal organization, and differentiation, and if manipulation of cholesterol/Cav-1/caveolae affects these responses. AIM 3: Test the activity of integrin- activated focal adhesion signaling pathways in MSCs within soft and stiff 3D substrates, and the regulatory effects of cholesterol/Cav-1/caveolae on focal adhesion signaling and downstream differentiation as influenced by soft and stiff 3D scaffolds. This will determine if focal adhesion signaling is involved in MSC substrate rigidity responses, and if such involvement is affected by cholesterol/Cav-1/caveolae.